Preparation Of Phospholipid Imprinted Materials Toward The Recognition And Application To Cell & Exosome

Phospholipids are the main components of cell membranes,and are classified into two major groups,glycerophospholipids and sphingolipids,depending on the phospholipid backbone.Phosphatidylserine(PS)belongs to the class of glycerophospholipids and is preferentially found in the inner leaflet of the plasma membrane.In addition,PS is found on the surface of apoptotic cells and exosomes,which are mainly regulated by aminophospholipid transferase.While sphingomyelin(SM)belongs to the class of sphingolipids,it is widely found in the outer leaflet of cell membranes.Phospholipids are amphiphilic,with a hydrophilic nitrogen-or phosphorus-containing head at one end and a lipophilic long hydrocarbon-based chain tail at the other end.The structural diversity of phospholipids is reflected in the complexity of their hydrophobic tails,so there are few methods for the specific recognition of lipids.Although a large number of antibodies and aptamers have been developed for the recognition of phospholipids,unfortunately they are often in limited supply,expensive and have poor storage durability,and these techniques are only applicable to free phospholipids,whereas selective targeting of cell membrane phospholipids is much more challenging and valuable.Recognition of the hydrophilic head of phospholipids is essential for cell surface phospholipid recognition based on the fact that the hydrophilic head of the phospholipid is exposed to the cell membrane.Molecularly imprinted polymers(MIPs)are used as artificial antibodies with low cost,chemical stability,and controllable imprinted cavities,which are capable of subject-object recognition with target molecules based on affinity.In this thesis,a novel imprinting strategy for phospholipids was explored to prepare molecularly imprinted polymers by combining a reverse-phase microemulsion system with targeted surface molecular imprinting.This strategy used PS and SM as template molecules for surface molecular imprinting on the hydrophilic heads of two phospholipids,resulting in phosphatidylserine-imprinted MIP(PS-imprinted MIP,PS-MIP)and sphingomyelinimprinted MIP(SM-imprinted MIP,SM-MIP)with high specificity and high selectivity.The main studies are described below:First,the core of the phospholipid imprinting strategy:(1)Surface imprinting of different hydrophilic heads of phospholipids in a reversed-phase microemulsion system,with regular directional distribution of phospholipids at the oil/water interface,which makes the imprinting of hydrophilic heads of phospholipids simple and overcomes the problem of difficult specific recognition due to the amphiphilic nature of phospholipids.(2)Phospholipids mainly rely on non-covalent interactions to bind to the imprinted polymers,and the application of silylating reagents allows the imprinting process to be precisely regulated,resulting in the preparation of phospholipid-imprinted polymers,which are characterized by selective recognition and high affinity.In this thesis,two phospholipids,PS and SM,were used as template molecules to prepare the imprinted polymers,which demonstrated the feasibility and general practicability of the method,and the phospholipid-imprinted polymers also showed good enrichment ability for free phospholipids in complex sample systems.Secondly,this thesis explored the selective recognition of specific phospholipids on the plasma membrane surface by PS-MIP and SM-MIP.It was found that PS-MIP could specifically recognize the marker PS on the plasma membrane of apoptotic cells,and could be applied in the field of apoptotic cell imaging.And the prepared SM-MIP can specifically recognize SM on the plasma membrane of cells with universal membrane recognition ability,which can be prepared as a drug delivery carrier.In this thesis,we further enhanced the SM blotting effect by optimizing the synthesis conditions of SM-MIP,and took advantage of its universal membrane recognition to design drug delivery carriers with the ability to penetrate the plasma membrane.Tenofovir(TFV),an antiviral drug,was selected for delivery with the aim of solving the problem of poor membrane permeability caused by its large polarity and difficulty in entering into the cell.In this thesis,molecularly imprinted materials with dualtemplated imprinted cavities(dt-MIP)were prepared with TFV and SM templates using a reverse-phase microemulsion system and a phospholipid surface imprinting strategy.The preparation of dt-MIP is characterized by good dispersion,large specific surface area,low cytotoxicity,separable by magnetic attraction and modifiable by fluorescent groups.The good plasma membrane SM-specific recognition ability of dt-MIP induced cellular cytosis,which in turn promoted the penetration of dt-MIP into the cell membrane.Quantitative analysis by LC-MS method revealed that dt-MIP could deliver TFV into the cell within 30 min,which improved the membrane permeability of TFV and had the effect of intracellular controlled release,minimizing the problem of high free drug concentration due to the premature release of TFV at undesired extracellular sites.Finally,exosomes are rich in biomarkers and are used in liquid biopsies for cancer diagnosis.Proteomic analysis of exosomes helps to identify cancer-related protein markers,but currently exosomes are detected in low abundance in complex body fluids and are susceptible to interference from heteroproteins,so it is crucial to isolate exosomes efficiently from complex body fluids.In this thesis,the feasibility of exosome enrichment by PS-MIP method was verified,and the ultracentrifugation method was used as a positive control.The results demonstrate that the PS-MIP method is characterized by simpler,faster,higher enrichment efficiency and fewer exosome residues,and that exosomes can be enriched from as low as 500 μL of urine or 5 μL of plasma.Quantitative analysis of proteins in exosomes by LC-MS and comparison with the exosome database demonstrated that the identified proteins were mainly derived from exosomes,and exosomes extracted from plasma and urine samples of patients with pancreatic cancer and healthy individuals were also analyzed proteomically.SLC9A3R1,SPAG9 and FTL were found to be overexpressed in patient urine exosomes,and EXTL2 and A2ML1 and PARK7 were overexpressed in patient plasma exosomes,which may be alternative biomarkers for pancreatic cancer diagnosis and prognostic assessment.In summary,the synthesized molecularly imprinted materials have great potential for selective property membrane recognition,targeted drug delivery and biomarker discovery.